The claimed invention relates generally to the fields of medicine and enzyme biochemistry. More particularly, the invention relates to compounds and methods for modulating the activity of Rho C.
The small GTPase family of proteins are central regulators of cell physiology. Five homologous subfamilies are found in the genomes of all eukaryotes; the S. cerevisiae genome includes 29 proteins in all five families, and the human genome encodes approximately 100 proteins. These five subfamilies have five overlapping but partially distinct functional roles. Ras family members regulate cell growth and division (A. Hall, Curr Opin Cell Biol (1993) 5(2):265-68; A. B. Vojtek et al., J Biol Chem (1998) 273(32):19925-28). Rho family members regulate cell motility, and shape through the actin skeleton (A. Hall, Science (1998) 279:509-14; D. J. Mackay et al., J Biol Chem (1998) 273:20685-88). ARF family members regulate cell adhesion and vesicle trafficking to and from the plasma membrane (A. L. Boman et al., Trends Biochem Sci (1995) 20(4):147-50; P. Chavrier et al., Curr Opin Cell Biol (1999) 11(4):466-75). Rab family members regulate intra-vesicular organelle trafficking (O. Martinez et al., Biochim Biophys Acta (1998) 1404(1-2):101-12; P. Chavrier et al., supra; F. Schimmoller et al., J Biol Chem (1998) 273(35):22161-14) and Ran family members regulate nuclear translocation and chromosomal segregation through regulation of microtubule assembly at the spindle pole (M. S. Moore, J Biol Chem (1998) 273(36):22857-60; M. G. Rush et al., Bioessays (1996) 18(2):103-12). These proteins stimulate other proteins in their GTP-bound state via physical interactions, and lose these associations and activities in the post-hydrolytic GDP-bound state. The hydrolysis reaction thus serves as molecular timer for the events triggered by the GTP-bound small G-protein. In addition, these GTPases also serve as signal integrators since the GTPases are regulated by other signaling pathway proteins; these signaling proteins are themselves regulated and promote or inhibit exchange of GDP for GTP or accelerate the GTP hydrolysis reaction. Ras was the first human small-GTPase to be appreciated in detail due to its identification as a human oncogene mutated in greater than 20% of human cancers (J. L. Bos, Cancer Res (1989) 49(17):4682-89). The ras mutants found in human cancers create a GTPase deficient form of ras which thus exists predominantly in the GTP bound-activated state.
The Rho family of small GTPase comprises more than ten members in humans and six members in yeast. In both organisms, control of the actin skeleton organization and localization is a major Rho function. The human Rho family is composed of three sub-families: Rho, Rac and CDC42 (K. Kaibuchi et al., Ann. Rev. Biochem. (1999) 68:459-86). These sub-families are all involved in control of the actin skeleton and cell adhesion. RhoA is the best-studied of RhoA-G group (collectively Rho) and has been closely associated with actin stress fiber formation in fibroblasts, and through its interaction with ROCK (Rho activated kinase) actin-myosin contraction leading to smooth muscle contraction. Yeast Rho1 is most homologous to human RhoA, and is found at the main site of organized actin in yeast (the bud), where it appears to regulate actin organization associated with budding. In addition, Rho1p controls cell wall bio-synthetic enzyme activity of 1,3-beta-D-glucan synthase (FKS1) during its physical association with the GTP-bound Rho1p. CDC42 and Rac-1 have also been well studied. CDC42 is closely associated with filopodia or microspike formation in fibroblasts and integrin activation. Rac-1 is a downstream component of the Ras signaling pathway from growth factor receptors and is closely associated with actin rearrangements leading to lamellipodia formation in fibroblasts (A. Hall, Science (1998) 279:509-14).
Rho proteins interact with several upstream and downstream components in signaling pathways that originate at the cell membrane with either G-protein coupled receptors, CDC42 and RhoA, or growth factor receptors, such as Rac-1.
The upstream pathways from membrane receptors to the Rho protein involves PI3-Kinase, PIP3, and a Db1-homology protein that is a PIP3 receptor and catalyzes guanine nucleo-tide exchange of Rho; it is thus termed a GEF (xe2x80x9cguanine nucleotide exchange factorxe2x80x9d). The GEFs for RhoA, and its close homologue RhoC, include Db1, Net1, Ost and Vav. These proteins all have Db1 homology domains (also known as RhoGEF domains) and pleckstrin homology domains, and all activate guanine nucleotide exchange by interaction with Rho proteins through their Db1-homology domain (R. A. Cerione et al., Curr Opin Cell Biol (1996) 8(2):216-22; I. P. Whitehead et al., Biochim Biophys Acta (1997) 1332(1):F1-23). The yeast upstream pathways from the cell membrane to Rho and beyond are highly related to those found in mammalian cells and include Tor2 (yeast PI3-kinase), and Rom1/Rom2 yeast Db1-homology and pleckstrin containing GEFs (K. Tanaka et al., Curr Opin Cell Biol (1998) 10(1):112-16). In both yeast and humans, Rho proteins are prenylated and associate, in their GDP bound states, with a guanine nucleotide-dissociation inhibitor (xe2x80x9cGDIxe2x80x9d). The GDI, known as RhoGDI in humans, and Rdi1p in yeast, solublizes the Rho protein and prevents its membrane association until activation by a GEF exchanges its GDP for GTP and allows its association with the membrane (T. K. Nomanbhoy et al., Biochemistry (1999) 38(6):1744-50; P. W. Read et al., Protein Sci (2000) 9(2):376-86).
The downstream pathways from Rho family members include many functionally and structurally homologous proteins. RhoA interacts with formin family members Dia1/Dia2, and yeast Rho1p interacts with Bni1 (another formin family member), while CDC42 interacts with WASP and WASP-N, a pair of proteins organized and regulated similarly to formin members. The formin family members have binding sites for the GTP-bound forms of Rho and also actin-nucleating domains whose exposure is controlled by binding of the GTP-Rho (N. Watanabe et al., Nat Cell Biol (1999) 1(3):136-43). In addition to formin interactions, Rho proteins interact with serine/threonine kinases. RhoA interacts with ROCK kinase, which then phosphorylates proteins that control actin polymerization; it also phosphorylates myosin regulators which control contraction in smooth muscles. Yeast Rho1p interacts with PKC1 which launches a MAP kinase cascade leading to control of transcription and the actin skeleton (S. B. Helliwell et al., Curr Biol (1998) 8(22):1211-14; K. Tanaka et al., Curr Opin Cell Biol (1998) 10(1):112-16).
The 3-dimensional structure of RhoA in its GTP, GDP and Mg2+ depleted states are known (K. Ihara et al., J Biol Chem (1998) 273(16):9656-66; R. Maesaki et al., Mol Cell (1999) 4(5):793-803; T. Shimizu et al., J Biol Chem (2000) 275(24):18311-17) as is the structure of RhoA-GTP in complex with an interaction domain of the downstream effector PKN (R. Maesaki et al., supra; R. Maesaki et al., J Struct Biol (1999) 126:166-70), and the structure of the complex of RhoA-GDP with Rho-GDI (K. Longenecker et al., Acta Crystallogr D Biol Crystallogr (1999) 55(Pt 9): 1503-15). The structure of RhoA in complex with rhoGAP is also known (K. Rittinger et al., Nature (1997) 388:693-97). The structure of these molecules combined with similarly detailed data regarding Ras and the Rho family member CDC42 yield consensus molecular mechanism for the GTPase function, GEF""s promotion of GDP exchange, GAP""s acceleration of GTPase activity and effector stimulation by RhoA-GTP. These studies show the guanine nucleotide bound in a pocket surrounded by three protein loops, known as switch region-I, switch region-II and the P-loop (the phosphate-binding loop). Switch region-I and -II interact extensively with GDI, GEF and effector domains in regions that occlude each other""s binding site. Switch region I and II are dramatically rearranged by GTP binding as compared to GDP-bound RhoA and this change exposes large new hydrophobic patches on the switch region surfaces. These newly exposed regions bind effectors.
The importance of Rho proteins in immune cell physiology is highlighted by the evolution of several different mechanisms to inactivate Rho-family proteins by pathogenic Clostridia species and other bacterial pathogens. These toxins are proteins that catalyze several different types of covalent modifications of the switch region-I of Rho proteins. This covalent modifications prevents the correct function of the Rho proteins (K. Aktories, Trends Microbiol (1997) 5(7):282-88; G. Schmidt et al., Naturwissenschaften (1998) 85(6):253-61). These toxins prevent leukocyte adhesion and diapeadisis and also reduce the production of some anti-bacterial metabolites; thus these toxins confer virulence to the strains possessing them.
Recent reports have shown an important role for Rho in cancer and metastasis. In colon, breast, and lung cancer, RhoA protein expression is elevated compared to the surrounding normal tissue. In breast cancer, RhoA, Rac and CDC42 are elevated. However, RhoA is the most dramatically elevated, and RhoA levels are correlated with disease severity (G. Fritz et al., Int J Cancer (1999) 81(5):682-87). In pancreatic cancers, RhoC mRNA levels are elevated in comparison to non-cancerous tissue, and the degree of RhoC elevation is positively correlated with clinical severity and negatively with patient survival. The RhoC elevated phenotype is closely associated with metastasis (H. Suwa et al., Br J Cancer (1998) 77(1):147-52). Furthermore, in mice, RhoA transformed-human tumor cells are more invasive than non-transformed cells (K. Yoshioka et al., Cancer Res (1999) 59(8):2004-10). Using in-vivo selection for mutations that cause increased metastatic potential in human melanoma cells injected into nude mice, it was recently shown that RhoC overexpression correlates with increased metastatic potential. Over-expression of RhoC from a retroviral vector by itself was sufficient to increase this potential (E. A. Clark et al., Nature (2000) 406:532-35). Thus, human cancers express elevated levels of Rho proteins and the degree of elevation correlates with disease severity and poor clinical prognosis.
The compelling case for the involvement of small G-protein in diseases has prompted a number of drug development attempts. Several different geranyl and farnesyl transferase inhibitors have been developed and several are now advanced in the clinic (N. E. Kohl, Ann NY Acad Sci (1999) 886:91-102). These inhibitors prevent farnesylation and/or geranylation of many proteins, including the small GTPases, and thus prevent their activity. These inhibitors have shown oral activity in animal models of Ras transformed tumorogenesis. Given their low selectivity and pan-prenylated protein specificity, the low toxicity and apparent high therapeutic ratio these compound have shown is surprising (D. W. End, Invest New Drugs (1999) 17(3):241-58; C. C. Kumar et al., Ann NY Acad Sci (1999) 886:122-31). An interesting approach to finding wild-type Ras inhibitors used electrospray mass spectrometry to detect compounds that form non-covalent complexes with Ras-GDP. A large library of compounds was screened and several hydroxylamine containing compounds that form complexes with the Mg2+ and the exterior lip of switch region-II were found; this binding site was mapped using NMR and a mass-spectrometric footprinting technique. The more avid of these compounds bind with affinities of 0.9 xcexcM (A. K. Ganguly et al., Bioorg Med Chem (1997) 5(5):817-20; A. K. Ganguly et al., Biochemistry (1998) 37(45):15631-37; A. G. Taveras et al., Bioorg Med Chem (1997) 5(1):125-33).
The GTPases present special problems as drug development targets. The difficulties arise due to the functions and properties of these proteins: they exist in numerous, but sometimes transient, complexes with partners (7-10 known partners for each protein). They exist inside most cells as GDP-bound enzymes in a complex with a GDI; their exchange to a GTP bound form happens instantaneously due to the high intracellular concentration of GTP (xcx9c1 mM) and their very high affinity for GTP. Their enzymatic activity and turnover is quite slow, and in a practical sense may happen only when a GAP associates with the protein. A specific issue related to discovery of mutant Ras inhibitors is that mutant-Ras forms found in tumors are GTPase deficient, and thus are GTP-bound. The Rho proteins are not locked into a single GTP-bound state, and therefore cycle between states. Thus Rho proteins may be easier targets to inhibit than mutant Ras has proven to be. However, these properties make it especially difficult to assay small G-proteins completely in in-vitro biochemical assays.
One aspect of the invention is a compound of formula 1 
wherein R1 is H, OH, or lower alkyl; R2, R3, R4, R5, and R6 are each independently H, halo, lower alkyl, OH, lower alkoxy, NH2, lower alkylamino, di(lower alkyl)amino, SH, lower alkylthio, NO2, or two residues together form a heterocyclic ring; R7, R8, R9, and R10 are each independently H, lower alkyl, OH, NH2, aryl, or aralkyl, where aryl and aralkyl are substituted with 0-3 moieties selected from the group consisting of halo, OH, NH2, lower alkyl, lower alkoxy, SH, lower alkylthio, and lower alkylamino; R11, R12, R13, R14, R15, and R16 are each independently H, halo, lower alkyl, OH, lower alkoxy, or NO2; and pharmaceutically acceptable salts thereof.
Another aspect of the invention is a method for inhibiting Rho C enzyme activity, by contacting said enzyme with a compound of formula 1.
Another aspect of the invention is a formulation for treating a disorder mediated by RhoC, comprising an effective amount of a compound of formula 1, and a pharmaceutically acceptable carrier.